PROJECT SUMMARY/ABSTRACT Defense against hypoglycemia is critical for survival, and is of particular importance in the clinically-significant setting of insulin-induced hypoglycemia. Insulin-induced hypoglycemia is prevalent in Type 1 and advanced Type 2 diabetes mellitus, and is associated with a far-ranging negative impact, including reduced work productivity and quality of life, decreased adherence to or recommendations for intensive insulin regimens, increased incidence of accidents and other morbidities, and occasionally death. While the body has developed a highly integrated defense system with which to prevent or correct hypoglycemia, in diabetes patients experiencing recurrent hypoglycemia due to over-insulinization, these counter-regulatory defenses are compromised, contributing to what can become a vicious cycle of hypoglycemia and often hypoglycemia unawareness (hypoglycemia-associated autonomic failure; HAAF) stemming from an abrogated sympathoadrenal arm of the counter-regulatory response. Several facets of ghrelin biology suggest that ghrelin may participate in the counter-regulatory response to insulin-induced hypoglycemia: 1) ghrelin secretion is directly stimulated by low glucose and sympathoadrenal activation; 2) the ensuing raised ghrelin has at its disposal many potential downstream targets with which to influence glucose handling, including interactions with several traditional counter-regulatory response hormones. However, while ghrelin regulation of blood glucose and by blood glucose have been evaluated in contexts such as caloric restriction, the overall role of the ghrelin system in the counter-regulatory response to insulin-induced hypoglycemia has not been fully assessed experimentally, for instance by re-creating insulin-induced hypoglycemia or HAAF in mice without an intact ghrelin system. Here, we will test the concept that ghrelin plays a key, protective, counter-regulatory role in the body?s response to insulin-induced hypoglycemia. In particular, we will dissociate the direct effects of insulin versus hypoglycemia on ghrelin secretion by performing insulin bolus-induced hypoglycemia tests, hyperinsulinemic-hypoglycemic clamps, and ex vivo ghrelin secretion studies in mice lacking insulin receptors selectively in ghrelin cells and phloridzin-hypoglycemic clamps in ghrelin-KO mice. We will determine requirement and sufficiency for GHSRs in AgRP neurons or SF1 neurons on the counter-regulatory response using Cre-lox transgenic mice and chemogenetic technology. Also, we will determine if ghrelin deletion increases susceptibility to HAAF by assessing the ghrelin response to hypoglycemia in wild-type mice with HAAF and the overall counter-regulatory response following recurrent hypoglycemia in mice lacking ghrelin. Our one-of-a-kind toolbox of recombinant mouse models targeting the ghrelin system, our team?s expertise in studying both ghrelin action and secretion, and our expertise in glucose metabolism including performing glucose clamps in mice will allow us to gain important and novel insights into not only hypoglycemia counter- regulation and the development of HAAF, but also ghrelin cell physiology and ghrelin action.